Isolated polynucleotides encoding d-arabino-3-hexulose-6-phosphate synthases from Methylophilus methylotrophus

ABSTRACT

The present invention provides polypeptides and polynucleotides involved in C1 assimilation in  Methylophilus methylotrophus  and methods of producing amino acids in microorganisms having enhanced or attenuated expression of these polypeptides and/or polynucleotides.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel polynucleotides encoding proteinsinvolved in one-carbon compounds metabolism, derived from microorganismsbelonging to methylotrophic bacteria and fragments thereof, polypeptidesencoded by the polynucleotides and fragments thereof, polynucleotidearrays comprising the polynucleotides and fragments thereof

2. Discussion of the Background

Amino acids such as L-lysine, L-glutamic acid, L-threonine, L-leucine,L-isoleucine, L-valine and L-phenylalanine are industrially produced byfermentation by using microorganisms that belong to the genusBrevibacterium, Corynebacterium, Bacillus, Escherichia, Streptomyces,Pseudomonas, Arthrobacter, Serratia, Penicillium, Candida or the like.In order to improve the productivity of amino acids, strains isolatedfrom nature or artificial mutants thereof have been used as thesemicroorganisms, various techniques have been disclosed for enhancingactivities of L-amino acid biosynthetic enzymes by using recombinant DNAtechniques, to increase the L-amino acid-producing ability.

Productivity of L-amino acids has been considerably increased bybreeding of microorganisms such as those mentioned above and theimprovement of production methods. However, in order to meet furtherincrease in the demand in future, development of methods for moreefficiently producing L-amino acids at lower cost have still beendesired.

As methods for producing amino acids by fermentation of methanol whichis a fermentation raw material available in a large amount at a lowcost, there have conventionally known methods using Achromobacter orPseudomonas microorganisms (Japanese Patent Publication (Kokoku) No.45-25273/1970), Protaminobacter microorganisms (Japanese PatentApplication Laid-open (Kokai) No. 49-125590/1974), Protaminobacter orMethanomonas microorganisms (Japanese Patent Application Laid-open(Kokai) No. 50-25790/1975), Microcyclus microorganisms (Japanese PatentApplication Laid-open (Kokai) No. 52-18886/1977), Methylobacillusmicroorganisms (Japanese Patent Application Laid-open (Kokai) No.4-91793/1992), Bacillus microorganisms (Japanese Patent ApplicationLaid-open (Kokai) No. 3-505284/1991) and others.

However, no methods have been described for producing L-amino acidsusing Methylophilus bacteria. Although methods described in EP 0 035 831A, EP 0 037 273 A and EP 0 066 994 A have been described as methods fortransforming Methylophilus bacteria using recombinant DNA, applyingrecombinant DNA techniques to improvement of amino acid productivity ofMethylophilus bacteria has not been described.

Therefore, prior to the present invention genes isolated fromMethylophilus bacteria that are involved in C1 assimilation and whichcan be used to improve the yield of amino acids in culturedmicroorganisms were not described.

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel measures for theimproved production of amino acids or an amino acid, where these aminoacids include asparagine, threonine, serine, glutamate, glycine,alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine,phenylalanine, histidine, lysine, tryptophan, arginine and the saltsthereof. In a preferred embodiment the amino acids are L-amino acids.

Such a process includes bacteria, which express a protein comprising anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40. In one embodimentthe polypeptides are encoded by a polynucleotide selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQID NO:39. In another embodiment the polypeptides are encoded by otherpolynucleotides which have substantial identity to the herein describedpolynucleotides or those which hybridize under stringent conditions.

Another object of the invention is to provide polynucleotide sequencesselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, and SEQ ID NO:39; as well as those polynucleotidesthat have substantial identity to these nucleotide sequences, preferablyat least 95% identity.

Another object of the invention is to provide isolated polypeptideshaving a sequence selected from the group consisting of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40; as well as thosepolypeptides that have substantial identity to these amino acidsequences, preferably at least 95% identity.

A further object of the invention is a method for producing a protein orproteins by culturing host cells containing the herein describedpolynucleotides under conditions and for a time suitable for expressionof the protein and collecting the protein produce.

Another object is the use of host cells having the polynucleotidesdescribed herein to produce amino acids , as well as use of suchisolated polypeptides in the production of amino acids.

Other objects of the invention include methods of detecting nucleic acidsequences homologous to at least one of: SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, and SEQ ID NO:39, particularly nucleic acidsequences encoding polypeptides that herein described proteins orpolypeptides and methods of making nucleic acids encoding suchpolypeptides.

The above objects highlight certain aspects of the invention. Additionalobjects, aspects and embodiments of the invention are found in thefollowing detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art of molecular biology. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed herein. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting.

Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition,Cold Spring Harbor Laboratory Press, New York (2001), Current Protocolsin Molecular Biology, Ausebel et al (eds.), John Wiley & Sons, New York(2001) and the various references cited therein.

Methylophilus methylotrophus (M. methylotrophus) is a gram negativeribulose monophosphate cycle methanol-utilizer, which can be used forthe large-scale production of a variety of fine chemicals includingamino acids, nucleic acids, vitamins, saccharides, and so on. Thepolynucleotides of this invention, therefore, can be used to identifymicroorganisms, which can be used to produce fine chemicals, forexample, by fermentative processes. Modulation of the expression of thepolynucleotides in the metabolism of one-carbon compounds of the presentinvention, can be used to modulate the production of one or more finechemicals from a microorganism (e.g., to improve the yield of productionof one or more fine chemicals from Methylophilus or Methylbacillusspecies).

The proteins encoded by the polynucleotides of the present invention arecapable of, for example, performing a function involved in themetabolism of one-carbon compounds in M. methylotrophus, such asmethanol, formaldehyde, formate, or methylamine. Given the availabilityof cloning vectors used in M. methylotrophus, such as those disclosed inMethane and Methanol Utilizers, Plenum Press, New York (1992) edited byJ. Colin Murrell and Howard Dalton, the nucleic acid molecules of thepresent invention may be used in the genetic engineering of thisorganism to make it better or more efficient producer of one or morefine chemicals.

There are a number of mechanisms by which the alteration of a protein ofthe present invention may affect the yield, production, and/orefficiency of production of a fine chemical from M. methylotrophusbacteria, which have the altered protein incorporated. Improving theability of the cell to utilize formaldehyde (e.g., by manipulating thegenes encoding enzymes involved in the incorporation and conversion ofthe compound into sugar compounds, such as fructose-6-phosphate), onemay increase the yield or productivity of desired fine chemicals.Furthermore, by suppressing the activity of enzymes involved in thewasteful pathway such as the conversion of formaldehyde to carbondioxide, one may also increase the yield or productivity of desired finechemicals.

“L-amino acids” or “amino acids” as used herein means one or more aminoacids, including their salts, preferably chosen from the following:L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine,L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine.

“Isolated” as used herein means separated out of its naturalenvironment.

“Polynucleotide” as used herein relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

“Polypeptides” as used herein are understood to mean peptides orproteins which comprise two or more amino acids bonded via peptidebonds. In particular, the term refers to polypeptides which are at least70%, preferably at least 80% and more preferably at least 90% to 95%identical to the polypeptides according to the present invention.Included within the scope of the present invention are polypeptidefragments of SEQ ID NO: 12 and SEQ ID NO: 14 or those which areidentical as described

Polynucleotides which encode the polypeptides of the invention as usedherein is understood to mean the sequences exemplified in thisapplication as well as those sequences which have substantial identityto SEQ ID NO: 11 and SEQ ID NO: 13 and which encode a molecule havingone or more of the bioactivities of the associated gene products.Preferably, such polynucleotides are those which are at least 70%,preferably at least 80% and more preferably at least 90% to 95%identical to SEQ ID NO: 11 and SEQ ID NO: 13.

Polynucleotides according to the invention may be employed as probes toisolate and/or identify RNA, cDNA and DNA molecules, e.g., full-lengthgenes or polynucleotides which code for the polypeptides describedherein. Likewise, the probes can be employed to isolate nucleic acids,polynucleotides or genes which have a high sequence similarity oridentity with the polynucleotides of the invention.

Polynucleotides of the invention may also be used to design primersuseful for the polymerase chain reaction to amplify, identify and/orisolate full-length DNA, RNA or other polynucleotides with high sequencehomology or identity to the polynucleotides of the invention, as wellas, polynucleotides that encode the polypeptides of the invention.Preferably, probes or primers are at least 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.Oligonucleotides with a length of at least 35, 40, 45, 50, 100, 150,200, 250 or 300 nucleotides may also be used.

Additionally, methods employing DNA chips, microarrays or similarrecombinant DNA technology that enables high throughput screening of DNAand polynucleotides that encode the herein described proteins orpolynucleotides with high sequence homology or identity to thepolynucleotides described herein. Such methods are known in the art andare described, for example, in Current Protocols in Molecular Biology,Ausebel et al (eds), John Wiley and Sons, Inc. New York (2000).

The polynucleotides and polypeptides of the present invention areinvolved in C1 assimilation in M. methylotrophus and include:

-   1. Phosphohexuloisomerse enzyme comprises the amino acid sequence of    SEQ ID NO:2 and is encoded by the phi gene which comprises the    polynucleotide SEQ ID NO:1;-   2. Methylene tetrahydromethanopterin tetrahydrofolate dehydrogenase    enzyme comprises the amino acid sequence of SEQ ID NO:4 and is    encoded by a mtdA gene which comprises the polynucleotide SEQ ID    NO:3;-   3. Methenyl H4MPT cyclohydrolase enzyme comprises the amino acid    sequence of SEQ ID NO:6 and is encoded by a mch gene which comprises    the polynucleotide SEQ ID NO:5;-   4. The D-arabino-3-hexulose 6-phosphate synthase enzymes: the hps2B    enzyme comprises the amino acid sequence of SEQ ID NO:8 and is    encoded by a hps2B gene comprising SEQ ID NO:7; the hps2A enzyme    comprises the amino acid sequence of SEQ ID NO:10 and is encoded by    a hps2A gene comprising SEQ ID NO:9; the hps1B enzyme comprises the    amino acid sequence of SEQ ID NO:12 and is encoded by a hps1B gene    comprising SEQ ID NO:11; and the hps1A enzyme comprises the amino    acid sequence of SEQ ID NO:14 and is encoded by a hps1A gene    comprising SEQ ID NO:13;-   5. The formylmethanofran dehydrogenase, chain C enzyme comprises the    amino acid sequence of SEQ ID NO:16 and is encoded by a fwdC gene    comprising SEQ ID NO:15;-   6. The formylmethanofran dehydrogenase, chain B enzyme comprises the    amino acid sequence of SEQ ID NO:18 and is encoded by a fwdB gene    comprising SEQ ID NO:17;-   7. The formylmethanofran dehydrogenase, chain A enzyme comprises the    amino acid sequence of SEQ ID NO:20 and is encoded by a fwdA gene    comprising SEQ ID NO:19;-   8. The methylenetetrahydrofolate    dehydrogenase/methylenyl-tetrahydrofolate cyclohydrolase enzyme    comprises the amino acid sequence of SEQ ID NO:22 and is encoded by    a foID gene comprising SEQ ID NO:21;-   9. The formyltetrahydrofolate synthetase enzyme comprises the amino    acid sequence of SEQ ID NO:24 and is encoded by a fhs gene    comprising SEQ ID NO:23;-   10. The formylmethanofuran-tetrahydromethanopterin formyltransferase    enzyme comprises the amino acid sequence of SEQ ID NO:26 and is    encoded by a ffsA gene comprising SEQ ID NO:25;-   11. The NAD-dependent formate dehydrogenase γ enzyme comprises the    amino acid sequence of SEQ ID NO:28 and is encoded by a fdhB gene    comprising SEQ ID NO:27;-   12. The NAD-dependent formate dehydrogenase δ enzyme comprises the    amino acid sequence of SEQ ID NO:30 and is encoded by a fdhD gene    comprising SEQ ID NO:29;-   13. The FdhC protein modulates the activity of formate dehydrogenase    and comprises the amino acid sequence of SEQ ID NO:32 and is encoded    by a polynucleotide comprising SEQ ID NO:31;-   14. The NAD-dependent formate dehydrogenase β enzyme comprises the    amino acid sequence of SEQ ID NO:34 and is encoded by a fdhB gene    comprising SEQ ID NO:33;-   15. The NAD-dependent formate dehydrogenase a enzyme comprises the    amino acid sequence of SEQ ID NO:36 and is encoded by a fdhA gene    comprising SEQ ID NO:35;-   16. The formaldehyde activated protein comprises the amino acid    sequence of SEQ ID NO:38 and is encoded by a fap gene comprising SEQ    ID NO:37;-   17. The glutathione-dependent formaldehyde enzyme comprises the    amino acid sequence of SEQ ID NO:40 and is encoded by a fad gene    comprising SEQ ID NO:39;

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing).

Typically, stringent conditions will be those in which the saltconcentration is less than approximately 1.5 M Na ion, typically about0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions also may be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (w/v; sodium dodecyl sulphate) at 37°C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodiumcitrate) at 50 to 55° C. Exemplary moderate stringency conditionsinclude hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37°C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1 M NaCl,1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA—DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl (Anal. Biochem., 138:267-284, 1984):Tm=81.5° C.+16.6 (log M)+0.41 (% GC)—0.61 (% form)—500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with approximately 90% identity are sought, the Tm can bedecreased 10° C.

Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (Tm); moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C. lower than the thermal melting point (Tm); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point (Tm). Using the equation,hybridization and wash compositions, and desired Tm, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

Stringent hybridization conditions are understood to mean thoseconditions where hybridization, either in solution or on a solidsupport, occur between two polynucleotide molecules which are 70% to100% homologous in nucleotide sequence which include 75%, 80%, 85%, 90%,95%, 98% and all values and subranges therebetween.

Homology, sequence similarity or sequence identity of nucleotide oramino acid sequences may be determined conventionally by using knownsoftware or computer programs. To find the best segment of identity orsimilarity of sequences, BLAST (Altschul et al (1990) J. Mol. Biol.215:403-410 and Lipman et al (1990) J. Mol. Biol. 215:403-410), FASTA(Lipman et al (1985) Science 227:1435-1441), or Smith and Waterman(Smith and Waterman (1981) J. Mol. Biol. 147:195-197) homology searchprograms can be used. To perform global alignments, sequence alignmentprograms such as the CLUSTAL W (Thompson et al (1994) Nucleic AcidsResearch 22:4673-4680) can be used.

The present invention also provides processes for preparing amino acidsusing bacteria that comprise at least one polynucleotide whoseexpression is enhanced or attenuated. Likewise, the invention alsoprovides processes for preparing amino acids using bacteria thatcomprise at least on polypeptide whose activity is enhanced orattenuated. Preferably, a bacterial cell with enhanced or attenuatedexpression of one or more of the polypeptides and/or polynucleotidesdescribed herein will improve amino acid yield at least 1% compared to abacterial strain not having the enhanced or attenuated expression. Forthe production of amino acids the M. methylotrophus polynucleotidesdescribed herein may be used to target expression, either by disruptionto turn off or increase or enhance the expression or relative activityof the polypeptide enzymes encoded therein.

The term “enhancement” as used herein means increasing intracellularactivity of one or more polypeptides in the bacterial cell, which inturn are encoded by the corresponding polynucleotides described herein.To facilitate such an increase, the copy number of the genescorresponding to the polynucleotides described herein may be increased.Alternatively, a strong and/or inducible promoter may be used to directthe expression of the polynucleotide, the polynucleotide being expressedeither as a transient expression vehicle or homologously orheterologously incorporated into the bacterial genome. In anotherembodiment, the promoter, regulatory region and/or the ribosome bindingsite upstream of the gene can be altered to achieve the over-expression.The expression may also be enhanced by increasing the relative half-lifeof the messenger RNA.

In another embodiment, the enzymatic activity of the polypeptide itselfmay be increased by employing one or more mutations in the polypeptideamino acid sequence, which increases the activity. For example, alteringthe relative Km of the polypeptide with its corresponding substrate willresult in enhanced activity. Likewise, the relative half-life of thepolypeptide may be increased.

In either scenario, that being enhanced gene expression or enhancedenzymatic activity, the enhancement may be achieved by altering thecomposition of the cell culture media and/or methods used for culturing.

“Enhanced expression” or “enhanced activity” as used herein means anincrease of at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or500% compared to a wild-type protein, polynucleotide, gene; or theactivity and/or the concentration of the protein present before thepolynucleotides or polypeptides are enhanced.

The term “attenuation” as used herein means a reduction or eliminationof the intracellular activity of the polypeptides in a bacterial cellthat are encoded by the corresponding polynucleotide. To facilitate suchan reduction or elimination, the copy number of the genes correspondingto the polynucleotides described herein may be decreased or removed.Alternatively, a weak and/or inducible promoter may used to direct theexpression of the polynucleotide, the polynucleotide being expressedeither as a transient expression vehicle or homologously orheterologously incorporated into the bacterial genome. For example, theendogenous promoter or regulatory region of the gene corresponding tothe isolated polynucleotides described herein may be replaced with theaforementioned weak and/or inducible promoter. Alternatively, thepromoter or regulatory region may be removed. The expression may also beattenuated by decreasing the relative half-life of the messenger RNA.

In another embodiment, the enzymatic activity of the polypeptide itselfmay be decreased or deleted by employing one or more mutations in thepolypeptide amino acid sequence, which decreases the activity or removesany detectable activity. For example, altering the relative Kd of thepolypeptide with its corresponding substrate will result in attenuatedactivity. Likewise, a decrease in the relative half-life of thepolypeptide will result in attenuated activity.

By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to25%, 0 to 10% or 0 to 5% of the activity or concentration of thewild-type protein or of the activity or concentration of the protein inthe starting microorganism.

Suitable vectors for carrying M. methylotrophus polynucleotides includethose vectors which can direct expression of the gene in bacterial cellsas known in the art. One embodiment of the present invention is wherebythe vectors contain an inducible or otherwise regulated expressionsystem whereby the M. methylotrophus polynucleotides may be expressedunder certain conditions and not expressed under other conditions.Furthermore, in another embodiment of the invention, the M.methylotrophus polynucleotides can be constitutively expressed. Examplesof such vectors and suitable cells in which they can be introduced aredescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001) and Current Protocols in Molecular Biology, Ausebel et al,(Eds.), John Wiley and Sons, Inc., New York, 2000.

Methods of introducing M. methylotrophus polynucleotides or vectorscontaining the M. methylotrophus polynucleotides includeelectroporation, conjugation, calcium-mediated transfection, infectionwith bacteriophage and other methods known in the art. These and othermethods are described in Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001) and Current Protocols in Molecular Biology,Ausebel et al, (Eds.), John Wiley and Sons, Inc., New York (2000).

The microorganisms that can be used in the present invention should havethe ability to produce amino acids, preferably L-amino acids, from asuitable carbon source, preferably carbon sources such as glucose,sucrose, lactose, fructose, maltose, molasses, starch, cellulose or fromglycerol and ethanol. The microorganisms can be Methylophilus bacteria,preferably Methylophilus methylotrophus, Escherichia bacteria,preferably Escherichia coli, Corynebacterium, preferably Corynebacteriumglutamicum.

Suitable culture conditions for the growth and/or production of M.methylotrophus polynucleotides are dependent on the cell type used.Likewise, culturing cells that contain attenuated or enhanced expressionof the M. methylotrophus polynucleotides or polypeptides, as describedherein, may be cultured in accordance with methods known in the art.Examples of culture conditions for various cells is described inSambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); CurrentProtocols in Molecular Biology, Ausebel et al, (Eds.), John Wiley andSons, Inc., 2000; and Cells: A Laboratory Manual (Vols. 1-3), Spector etal, (Eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., (1988).

Following culturing the polypeptide or protein products, which areencoded by the M. methylotrophus polynucleotides, may be purified usingknown methods of protein purification. These methods include highperformance liquid chromatography (HPLC), ion-exchange chromatography,size exclusion chromatography; affinity separations using materials suchas beads with exposed heparin, metals, or lipids; or other approachesknown to those skilled in the art. These and other methods of proteinpurification are disclosed in Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001); Current Protocols in Molecular Biology,Ausebel et al, eds., John Wiley and Sons, Inc., 2000 and ProteinPurification, Scopes and Cantor, (Eds.), Springer-Verlag, (1994).Likewise, the amino acids produced may be purified by methods known inthe art using similar chromatography devices.

The invention also provide antibodies that bind to the polypeptides ofthe present invention. Antibodies binding to the polypeptides can beeither monoclonal or polyclonal, preferably the antibodies aremonoclonal. Methods for obtaining antibodies that bind to thepolypeptides are known in the art and are described, for example, inHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988).

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting unless otherwise specified.

EXAMPLES

Whole genome sequencing using random shotgun method is described inFleischman R. D. et. al. (1995) Science, 269: 496-512.

Example 1 Construction of Genomic Libraries of Methylophilusmethylotrophus

M. methylotrophus AS1 was cultured at 30° C. in the 121 medium describedin the Catalogue of Strains (The National Collections of Industrial andMarine Bacteria Ltd., 1994). Cells were collected by centrifugation.Genomic DNA was isolated using the Genome-tip system (Qiagen K.K.,Tokyo, Japan). The genomic DNA was sheared and fragmentized bysonication. The resultant fragments in the 1- to 2-kb size range werepurified by gel electrophoresis through 1% low-melting agarose gel,followed by recovery using the Wizard DNA purification kit (Promega KK,Tokyo, Japan). The recovered fragments were ligated to the high-copynumber vector pUC118 treated by Hincli and bacterial alkalinephosphatase (Takara Shuzo, Kyoto, Japan), and this was designated pUC118library.

For larger fragments (9- to 11-kb in size), the genomic DNA waspartially digested by restriction endonuclease Sau3AI, followed by 0.6%agarose gel electrophoresis. The DNA fragments corresponding 9-kb to11-kb in size were excised from gel and were recovered using the DNACELL(Daiichi Pure Chemicals, Tokyo, Japan). The recovered fragments wereligated into the low-coy number vector pMW118 (Nippon Gene, Toyama,Japan), which is a derivative of the pSC101 (Bernaidi, A. and Bernardi,F. (1984) Nucleic Acids Res. 12, 9415-9426). This library composed oflarge DNA fragments was designated pMW118 library.

General DNA manipulation was performed according to previously describedmethods (Sambrook et. al. (1989) “Molecular Cloning: A LaboratoryManual/Second Edition”, Cold Spring Harbor Laboratory Press).

Example 2 DNA Sequencing and Sequence Assembly

The pUC118 library were transformed into Escherichia coli DH5a andplated on Luria-Bertani medium containing 100 μg/ml ampicillin and 40μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal). The whitecolonies were picked up and cultured in Luria-Bertani medium containing100 μg/ml ampicillin. The individual colony was cultured in the well ofthe 96 deep-well plates, and the plasmids were isolated using QIAprepTurbo Kit (Qiagen). The DNA fragments inserted into pUC18 were sequencedusing a M13 reverse primer. The shotgun sequencing was performed withthe BigDye terminators and 3700 DNA analyzer (Applied Biosystems Japan,Tokyo, Japan). Approximately 50,000 samples from pUC18 librarycorresponding to coverage of approximately 8-fold to the genome sizewere analyzed and the sequences were assembled by Phred/Phrap software(CodonCode, MA, USA). This assembly treatment yielded 60 contigs withmore than 5 kb in size.

As for pMW library, 2,000 clones corresponding to coverage ofapproximately 5-fold were sequenced using both M13 forward and reverseprimers. The end-sequence data were analyzed and the linking clonesbetween contigs were selected from pMW118 library. The insertedfragments of selected clones were amplified by the polymerase chainreaction (PCR) using LA Taq polymerase (Takara Shuzo) and M.methylotrophus genomic DNA as a template. These products of PCR wereentirely sequenced as described in Example 1, and the gap DNA sequencesbetween contigs were determined. By the additional sequence information,the Phrap assembly software reduced the number of contigs with more than5 kb in size to 24. Then the 48 DNA primers with sequences complementaryto the end-sequences of the 24 contigs were prepared. All possiblepairwise combination of the primers were tested by PCR to amplify theDNA fragments of M. methylotrophus genomic DNA. The amplified productswere sequenced directly. In several cases, the additional primerscomplementary to different sequences at the end of the contig were used.This strategy could close all of the remaining physical gaps andresulted in a single circular contig. Several regions that had beensequenced in only one direction and had postulated secondary structurewere confirmed. By this research, the genome of M. methylotrophus wasfound to be a single circular with the size of 2,869,603 bases and GCcontent of 49.6%.

Example 3 Sequence Analysis and Annotation

Sequence analysis and annotation was managed using the Genome Gamblersoftware (Sakiyama, T. et. al. (2000) Biosci. Biotechnol. Biochem. 64:670-673). All open reading frames of more than 150 bp in length wereextracted and the translated amino acid sequences were searched againstnon-redundant protein sequences in GenBank using the BLAST program(Altschul, S. F. et. al. (1990) J. Mol. Biol. 215, 403-410). Of putativepolynucleotide encoding sequences with significant similarities to thesequences in public databases (BLASTP scores of more than 100), thegenes involved in methanol metabolism were selected. Start codons (AUGor GUG) were putatively identified by similarity of the genes and theirproximity to the ribosome binding sequences (Shine, J. and Dalgamo, L.(1975) Eur. J. Biochem. 57: 221-230). Careful assignment of genefunction resulted in the identification of the formaldehydedehydrogenase gene (fadH), the formate dehydrogenase complex genes(fdhGBACD). The two key enzymes of the ribulose monophosphate pathway,D-arabino-3-hexulose 6-phosphate synthase (hps1A) andphosphohexuloisomerase (phi) were found probably in operon, however,three other hps-like genes (hps1B, hps2A, and hps2B) were identifiedindependently. The one-carbon unit (C1) transfer enzymes found inMethylobacterium extorquens, formaldehyde-activating enzyme (fap)(Vorholt, J. A. (2000) J. Bacteriol. 182, 6645-6650), methylenetetrahydromethanopterin dehydrogenase (mtdA), methenyltetrahydromethanopterin cyclohydrolase gene (mch),formylmethanofuran-tetrahydromethanopterin N-formyltransferase gene(ffsA), and formylmethanofran dehydrogenase subunit genes A, B, and C(fwdBA and fwdC) (Chistoserdova L, et. al. (1998) Science 281, 99-102)were identified in this organism. The bifunctional enzyme,methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolatecyclohydrolase gene (folD) involved in C1 transfer via tetrahydrofolatewas also identified.

Obviously, numerous modifications and variations on the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. An isolated polynucleotide, which encodes a protein comprising theamino acid sequence selected from the group consisting of SEQ ID NO:12and SEQ ID NO:14.
 2. A vector comprising at least one isolatedpolynucleotide of claim
 1. 3. A host cell transformed with at least oneisolated polynucleotide of claim
 1. 4. The host cell of claim 3, whichis a Methylophilus bacterium.
 5. The host cell of claim 4, which is aMethylophilus methylotrophus bacterium.
 6. A method of making a proteincomprising: culturing the host cell of claim 3 for a time and underconditions suitable for expression of said protein; and collecting saidprotein.
 7. An isolated polynucleotide, which comprises a nucleotidesequence selected from the group consisting of SEQ ID NO:11 and SEQ IDNO:13.
 8. A vector comprising at least one isolated polynucleotide ofclaim
 7. 9. A host cell transformed with the at least one isolatedpolynucleotide of claim
 7. 10. The host cell of claim 9, which is aMethylophilus bacterium.
 11. The host cell of claim 10, which is aMethylophilus methylotrophus bacterium.
 12. A method of making a proteincomprising: culturing the host cell of claim 9 for a time and underconditions suitable for the expression of the polynucleotide to producea protein; and collecting said protein.
 13. An isolated polynucleotide,which hybridizes under high stringent conditions to at least one of theisolated polynucleotides of claim 7, wherein said polynucleotide encodesa protein having the activity of D-arabino-3-hexulose 6-phosphatesynthase.
 14. A vector comprising the isolated polynucleotide of claim13.
 15. A host cell transformed with the isolated polynucleotide ofclaim
 13. 16. A method of making a protein comprising: culturing thehost cell of claim 15 for a time and under conditions suitable for theexpression of the polynucleotide to produce a protein; and collectingsaid protein.
 17. An isolated polynucleotide, which is at least 95%identical to the polynucleotide of claim 7, and wherein saidpolynucleotide encodes a protein having the activity ofD-arabino-3-hexulose 6-phosphate synthase.
 18. A vector comprising theisolated polynucleotide of claim
 17. 19. A host cell transformed withthe isolated polynucleotide of claim
 17. 20. A method of making aprotein comprising: culturing the host cell of claim 19 for a time andunder conditions suitable for the expression of the polynucleotide toproduce a protein; and collecting said protein.